The characterization of the structure and function of proteins is of critical importance in the biotechnology industry. As such, innovative methods are continually being developed to carry out this task. This article gives an introduction to the field of proteins, their structure and composition, and how molecular weight, size and zeta potential measurements can be applied.
Figure 1. Amino acid structure.
Amino acids are tiny molecules with a common structure. They have a central carbon atom attached to a hydrogen atom, an amino and a carboxyl group, and a fourth functional group (R), which is variable.
Figure 1 shows the basic structure of amino acids in both its charged and uncharged states.
Figure 2. Peptide bond formation.
Amino acids attach to one another through bonds called peptide bonds between the amino nitrogen and the carboxyl carbon. When the bond is formed, a water molecule is released, as shown in Figure 2.
Using these peptide bonds, amino acids can join together in chains of nearly any sequence, which are known as polypeptides. When a polypeptide is of an appropriate size, structure and sequence, it functionally becomes a protein.
A protein’s function is ascertained by its structure rather than its sequence of amino acids. However, the sequence of the amino acids is important for determining the end structure of the protein. Functional proteins exhibit a tightly regulated structure, which is held together by hydrophobic interactions, hydrogen bonds and the Van der Waals forces between nearby amino acids, as well as di-sulphide bridges between cysteine residues. The protein structure has four levels of complexity.
The primary structure describes the chain of amino acids in the polypeptide chain. The secondary structure describes the huge regular sub-structures. The α-helix and the β-sheet are two major sub-structures that form as secondary structure.
Figure 3 shows α-helical structure.
Figure 3. α-helical structures.
Tertiary structure, which forms from the secondary structures, is the final 3D structure of the protein. It is held together by hydrophobic interactions, disulphide bridges, and hydrogen bonds and the Van der Waals forces.
Figure 4. Ribbon diagram of the structure of hemoglobin.
Conversely, some proteins can only function when two or more polypeptide chains form dimmers or trimers, which are known as oligomers. The arrangement made by the formation of oligomers is called the quaternary structure.
For example, the homotetramer is the final structure of hemoglobin and contains four sub-units, each itself heterotetrameric comprising two pairs of sub-units α and β (Figure 4).
Translation refers to the process of manufacturing a protein within a biological cell. At this stage, amino acids are linked together sequentially and the protein folds. This process is carried out by ribosomes, which are partially protein and convert the sequence of a strand of RNA into a sequence of amino acids for a protein.
As temperature increases, the internal forces holding the protein structure are overcome and the protein unfolds. Changes in pH will not only affect the di-sulphide bridge formation but will also impact the ionization state of the various functional groups on the amino acids which could be involved in internal bonds. In most proteins, this process continues until the protein structure is entirely lost, including the protein activity. This protein is said to be denatured.
As a protein begins to denature, dipoles hydrophobic regions and charged ionic groups will be exposed to the surrounding medium. Partially denatured proteins bind to each other through these regions and the same forces which hold the structure of the protein together will hold proteins to each other. These strong bonds can hold many proteins together in groups and do not dissociate without completely denaturing the proteins. This process is known as aggregation.
Aggregation must be prevented during the storage or production of a therapeutic protein as aggregates in pharmaceutical preparations can cause strong immune responses in patients.
Proteins act as transporters for ions or other molecules and regulate most chemical reactions which occur within living organisms. They signal between and within cells locally and throughout an organism. Proteins also build and breakdown DNA and other proteins; as well as stimulating and regulating cell growth and division.
To refer to a previous example, oxygen is carried by hemoglobin in the blood. When an oxygen molecule binds to hemoglobin, it causes the structure of the protein to change. This is known as conformational change, which is a common process in protein activity.
Figure 5. Antibody structure.
Antibodies or immunoglobulins are large proteins that have a defined structure (Figure 5). The base of the molecule has the same primary, secondary and tertiary structure within a given organism. Antibodies are made to bind to antigens or foreign objects, such as viruses or bacteria within the body. Antibodies have a large number of variants in order to identify as many foreign molecules as possible. They are widely used in biotechnology laboratories.
Batch Dynamic Light Scattering
Size measurement is the basic measurement of proteins which can be carried out with batch dynamic light scattering (DLS). The Zetasizer software has a model to predict the protein’s molecular weight from its hydrodynamic size by DLS. The function and activity of a protein is directly related to proper folding and structure.
As such, activity is also directly associated with the size of the protein. Therefore, size can also be used as a predictor of activity. In order to function appropriately, many proteins depend on a correct quaternary structure. This means, hydrodynamic size can also be used as a predictor of activity.
Light scattering techniques are especially sensitive to larger molecules in the preparation of smaller molecules. Aggregate formation will result in the increase in the size of a protein. The sensitivity of the DLS measurement to larger proteins means that the initial stages of denaturation will promote changes in the mean hydrodynamic size. Therefore, DLS is the most sensitive method for detecting trace quantities of aggregates in preparations.
Static Light Scattering
Static light scattering (SLS) measurements can also be made on proteins. As long as the concentrations are precisely known, many protein samples can be applied for batch measurements of molecular weight using SLS. By determining the amount of light scattered at different concentrations of the sample, the molecular weight can be determined by producing a Debye plot.
A high-sensitive instrument such as the Zetasizer Nano ZSP and the patented diffusion barrier technique can be used to perform zeta-potential measurements of proteins. Overall, zeta-potential is a measure of the strength of the repulsive forces between molecules in solution.
Traditionally, this has been used as a primary indicator of the stability of a sample preparation. With a high zeta-potential and a high intermolecular repulsive force, a protein or drug preparation can be anticipated to be stable for longer period of time when compared to a similar preparation with low zeta-potential.
Figure 6. A: Zetasizer Nano B: Zetasizer APS C: Zetasizer µV D: GPCmax.
Zetasizer Nano (Figure 6A) is the most versatile light scattering instrument available on the market. The Zetasizer Nano ZSP has been particularly designed to deliver the sensitivity demanded for zeta potential and size measurement of poorly scattering materials such as proteins.
In high-throughput situations, where screening is performed for possible drug candidates, the Zetasizer APS with DLS plate sampling technology (Figure 6B) provides a suitable solution. It is the most sensitive and time-efficient DLS instrument on the market.
In protein analysis studies, proteins are usually purified and are available in very small quantities. In such situations, the Zetasizer µV (Figure 6C) delivers the proven sensitivity of the Zetasizer range using volumes down to 2µl. In addition, the Viscotek range of detectors (Figure 6D) provides precise and complete information on the molecular size, weight and structure of proteins.
This information has been sourced, reviewed and adapted from materials provided by Malvern Panalytical.
For more information on this source, please visit Malvern Panalytical.